Tiotropium Attenuates Virus-Induced Pulmonary Inflammation in Cigarette Smoke-Exposed Mice.

Viral infections trigger exacerbations in chronic obstructive pulmonary disease (COPD), and tiotropium, a M3 receptor antagonist, reduces exacerbations in patients by unknown mechanisms. In this report, we investigated whether tiotropium has anti-inflammatory effects in mice exposed to cigarette smoke (CS) and infected with influenza virus A/PR/8/34 (H1N1) or respiratory syncytial virus (RSV) and compared these effects with those of steroid fluticasone and PDE4-inhibitor roflumilast. Mice were exposed to CS; infected with H1N1 or RSV; and treated with tiotropium, fluticasone, or roflumilast. The amount of cells and cytokine levels in the airways, lung function, and viral load was determined. NCI-H292 cells were infected with H1N1 or RSV and treated with the drugs. In CS/H1N1-exposed mice, tiotropium reduced neutrophil and macrophage numbers and levels of interleukin-6 (IL-6) and interferon-γ (IFN-γ) in the airways and improved lung function. In contrast, fluticasone increased the loss of body weight; failed to reduce neutrophil or macrophage numbers; increased IL-6, KC, and tumor necrosis factor-α (TNF-α) in the lungs; and worsened lung function. Treatment with roflumilast reduced macrophage numbers, IL-6, and KC in the lungs but had no effect on neutrophil numbers or lung function. In CS/RSV-exposed mice, treatment with tiotropium, but not fluticasone or roflumilast, reduced neutrophil numbers and IL-6 and TNF-α levels in the lungs. Viral load of H1N1 and RSV was significantly elevated in CS/virus-exposed mice and NCI-H292 cells after fluticasone treatment, whereas tiotropium and roflumilast had no effect. In conclusion, tiotropium has anti-inflammatory effects on CS/virus-induced inflammation in mice that are superior to the effects of roflumilast and fluticasone. This finding might help to explain the observed reduction of exacerbation rates in COPD patients.

Modeling Pulmonary Disease Pathways Using Recombinant Adeno-Associated Virus 6.2.

Viral vectors have been applied successfully to generate disease-related animal models and to functionally characterize target genes in vivo. However, broader application is still limited by complex vector production, biosafety requirements, and vector-mediated immunogenic responses, possibly interfering with disease-relevant pathways. Here, we describe adeno-associated virus (AAV) variant 6.2 as an ideal vector for lung delivery in mice, overcoming most of the aforementioned limitations. In a proof-of-concept study using AAV6.2 vectors expressing IL-13 and transforming growth factor-ß1 (TGF-ß1), we were able to induce hallmarks of severe asthma and pulmonary fibrosis, respectively. Phenotypic characterization and deep sequencing analysis of the AAV-IL-13 asthma model revealed a characteristic disease signature. Furthermore, suitability of the model for compound testing was also demonstrated by pharmacological intervention studies using an anti-IL-13 antibody and dexamethasone. Similarly, the AAV-TGF-ß1 fibrosis model showed several disease-like pathophenotypes monitored by micro-computed tomography imaging and lung function measurement. Most importantly, analyses using stuffer control vectors demonstrated that in contrast to a common adenovirus-5 vector, AAV6.2 vectors did not induce any measurable inflammation and therefore carry a lower risk of altering relevant readouts. In conclusion, we propose AAV6.2 as an ideal vector system for the functional characterization of target genes in the context of pulmonary diseases in mice.

The long-acting ß2 -adrenoceptor agonist olodaterol attenuates pulmonary inflammation.

BACKGROUND AND PURPOSE: ß2 -adrenoceptor agonists are widely used in the management of obstructive airway diseases. Besides their bronchodilatory effect, several studies suggest inhibitory effects on various aspects of inflammation. The aim of our study was to determine the efficacy of the long-acting ß2 -adrenoceptor agonist olodaterol to inhibit pulmonary inflammation and to elucidate mechanism(s) underlying its anti-inflammatory actions. EXPERIMENTAL APPROACH: Olodaterol was tested in murine and guinea pig models of cigarette smoke- and LPS-induced lung inflammation. Furthermore, effects of olodaterol on the LPS-induced pro-inflammatory mediator release from human parenchymal explants, CD11b adhesion molecule expression on human granulocytes TNF-α release from human whole blood and on the IL-8-induced migration of human peripheral blood neutrophils were investigated. KEY RESULTS: Olodaterol dose-dependently attenuated cell influx and pro-inflammatory mediator release in murine and guinea pig models of pulmonary inflammation. These anti-inflammatory effects were observed at doses relevant to their bronchodilatory efficacy. Mechanistically, olodaterol attenuated pro-inflammatory mediator release from human parenchymal explants and whole blood and reduced expression of CD11b adhesion molecules on granulocytes, but without direct effects on IL-8-induced neutrophil transwell migration. CONCLUSIONS AND IMPLICATIONS: This is the first evidence for the anti-inflammatory efficacy of a ß2 -adrenoceptor agonist in models of lung inflammation induced by cigarette smoke. The long-acting ß2 -adrenoceptor agonist olodaterol attenuated pulmonary inflammation through mechanisms that are separate from direct inhibition of bronchoconstriction. Furthermore, the in vivo data suggest that the anti-inflammatory properties of olodaterol are maintained after repeated dosing for 4 days.

Development of a novel severe triple allergen asthma model in mice which is resistant to dexamethasone and partially resistant to TLR7 and TLR9 agonist treatment.

Severe asthma is characterised by persistent inflammation, hyperreactivity and remodeling of the airways. No efficient treatment is available, this is particularly the case for steroid resistant phenotypes. Our aim therefore was to develop a preclinical model showing characteristics of severe human asthma including steroid insensitivity. Mice were first sensitized with ovalbumin, extracts of cockroach or house dust mite followed by a challenge period of seven weeks. Further to this, an additional group of mice was sensitized with all three allergens and then challenged with allergen alternating weekly between allergens. All three allergens applied separately to the mice induced comparably strong Th2-type airway inflammation, airway hyperreactivity and airway remodeling, which was characterised by fibrosis and increased smooth muscle thickness. In contrast, application of all three allergens together resulted in a greater Th2 response and increased airway hyperreactivity and a stronger albeit not significant remodeling phenotype compared to using HDM or CRA. In this triple allergen model dexamethasone application, during the last 4 weeks of challenge, showed no suppressive effects on any of these parameters in this model. In contrast, both TLR7 agonist resiquimod and TLR9 agonist CpG-ODN reduced allergen-specific IgE, eosinophils, and collagen I in the lungs. The TLR9 agonist also reduced IL-4 and IL-5 whilst increasing IFN-γ and strongly IL-10 levels in the lungs, effects not seen with the TLR7 agonist. However, neither TLR agonist had any effect on airway hyperreactivity and airway smooth muscle mass. In conclusion we have developed a severe asthma model, which is steroid resistant and only partially sensitive to TLR7 and TLR9 agonist treatment. This model may be particular useful to test new potential therapeutics aiming at treating steroid resistant asthma in humans and investigating the underlying mechanisms responsible for steroid insensitivity.

Induced Syk deletion leads to suppressed allergic responses but has no effect on neutrophil or monocyte migration in vivo.

The spleen tyrosine kinase (Syk) is a key mediator of immunoreceptor signaling in immune cells. Thus, interfering with the function of Syk by genetic deletion or pharmacological inhibition might influence a variety of allergic and autoimmune processes. Since conventional Syk knockout mice are not viable, studies addressing the effect of Syk deletion in adult animals have been limited. To further explore functions of Syk in animal models of allergy and to shed light on the role of Syk in the in vivo migration of neutrophils and monocytes, we generated inducible Syk knockout mice. These mice harbor a floxed Syk gene and a tamoxifen-inducible Cre recombinase under the control of the ubiquitously active Rosa26-promoter. Thus, treatment of mice with tamoxifen leads to the deletion of Syk in all organs. Syk-deleted mice were analyzed in mast cell-dependent models and in models focusing on neutrophil and monocyte migration. We show that Syk deletion in adult mice reduces inflammatory responses in mast cell-driven animal models of allergy and asthma but has no effect on the migration of neutrophils and monocytes. Therefore, the inducible Syk knockout mice presented here provide a valuable tool to further explore the role of Syk in disease-related animal models.

TLR agonist mediated suppression of allergic responses is associated with increased innate inflammation in the airways.

Toll-like receptor (TLR) mediated signaling induces pro-inflammatory responses and can both suppress and exacerbate allergic responses in the airways. The aim of our study was to directly compare the efficacy of different TLR agonists in inhibiting or exacerbating the development of Th2-mediated responses in the airways and investigate if the suppressive effects were associated with increased pro-inflammatory responses. Mice were immunized on day 0, 14 and 21 by intraperitoneal injection of ovalbumin/alum and exposed to ovalbumin aerosol on day 26 and 27. TLR2, TLR3, TLR4, TLR7 and TLR9 agonists (0.001, 0.01, 0.1, or 1 mg/kg) were administered intratracheally 1 h before each allergen exposure. Both the TLR7 and TLR9 agonists dose dependently reduced airway eosinophilia, while the TLR3 agonist only reduced airway eosinophilia at a dose of 1.0 mg/kg. The TLR2 and TLR4 agonists potentiated eosinophilia. All TLR agonists enhanced neutrophil numbers at doses as low as 0.01 mg/kg, in particular TLR2 and TLR4 agonists. TLR7 and TLR9 agonists also significantly reduced IL-4 and IL-5 levels and all TLR agonists, with the exception of TLR7, enhanced the amount IL-1ß, IL-6, and TNF-α detected in the whole lung lavage. Only application of TLR9 agonist induced detectable levels of IL-10 in the lung. Suppressive effects of the TLR agonists were not dependent upon IFN-γ and IL-10 or associated with increased numbers of Foxp3(+)CD4(+) Tr cells in the lavage fluid. Airway resistance was reduced significantly only when TLR7 agonist was administered. When applied therapeutically 2 days after allergen exposure, all TLR agonists, except TLR2, similarly reduced airway eosinophilia and IL-4 levels. Taken together our results show that TLR7 agonists had the strongest anti-asthmatic effects with the lowest pro-inflammatory potential, suggesting that activating TLR7 may have the greatest potential to treat allergic disorders in humans.